Dairy cows are significant investments for dairy farmers, and enormous efforts, such as animal breeding and artificial insemination, have been and continue to be made to ensure that the animals have high and sustained productivity, and that the milk produced is of high quality. The dairy cattle genome has been significantly restructured over the past 30 years due to intensive selection for production traits.
Reproductive deterioration in high-producing dairy cows has caused substantial economic loss in the dairy cattle industry (Lucy, 2007). Two of the key factors contributing to decreasing fertility of dairy cow are low fertilization rate and early embryonic mortality (Royal et al., 2000. Sheldon et al. 2006), both of which occur during the pre-implantation embryo development. Although genetic factors are known to affect reproductive performance (Shook, 2006), the identification of specific genes has been a challenge, probably due to the low accuracy of fertility data collected in the field, and the low heritability of these traits (VanRaden et al., 2004). Thus, understanding the genetic regulation of bovine pre-implantation embryo development and identifying associated biomarkers are becoming progressively essential to improving dairy cattle fertility.
To investigate the relationship between genetic factors and pre-implantation embryo development in bovine, the present inventor has created an in vitro fertilization (IVF) system, which enables the identification of genetic factors affecting fertilization and early embryonic development at both the cow and embryo levels. At the cow level, the IVF system has been utilized to uncover associations of cow genotypes with fertilization success and blastocyst rate of embryos produced from these cows (Driver et al. 2009; Huang et al. 2010a; Khatib et al. 2009a,b; Khatib et al. 2008a,b; Wang et al. 2009). At the embryo level, differential gene expression between normal blastocysts and degenerate embryos has been investigated by applying RNA-Seq (Huang & Khatib, 2010), microarray expression (Huang et al., 2010b), and candidate gene and pathway analyses (Laporta et al., 2011; Zhang et al., 2011).
The transforming growth factor-β (TGF-β) signaling pathway has long been acknowledged for signal transduction and other intracellular activities, such as cell division, differentiation, migration, apoptosis, and transformation (Santibanez et al., 2011). In addition, several studies have suggested the involvement of the TGF-β signaling cascade and its components in preimplantation embryo development as well as ovarian function (Shimasaki et al., 2004; Zhang et al., 2007). For example, Said et al. (2010) recently reported that known members of TGF-β pathway showed dynamic changes in gene expression profiles during the three early stages of human embryonic development, including oocytes, day-3 embryos, and human embryonic stem cells on day 7. Also, results from the mouse showed that multiple bone morphogenetic proteins (BMPs) and SMAD6 in the TGF-β pathway were found to be expressed in a stage-specific pattern and were developmentally regulated in oocytes and preimplantation embryos (Wang et al., 2004). BMPs and GDF9, also a member of TGF-β, have been reported as crucial regulators of folliculogenesis in mouse models (Otsuka et al. 2011; Trombly et al. 2009).
It is worth mentioning that most of the reported data on TGF-β pathway genes have been generated in the mouse model and there is little information on other species such as cattle. Interestingly, in a recent transcriptomic study of the bovine IVF system, the TGF-β pathway was found to be upregulated in degenerate embryos compared to blastocysts using microarrays (Huang et al., 2010b). However, several genes from this pathway were not included in the microarray analysis.
Many components of the signaling cascade of TGF-β pathway are known, and they include ligands (BMP4, GDF9, and INHBA); receptors (BMPR1A and ACVR1); SMAD proteins (SMAD2); upstream regulators (THBS2, THBS4, DCN); and downstream regulators (ID3, BMPER, PPP2R1A, RPS6KB2, PITX2). The signaling process of this pathway necessitates coordination of gene regulation among the different members of the pathway. For example, the activation of latent TGF-β requires binding of the thrombospondin-1 (THBS1) to the TGF-β precursor complex (Murphy-Ullrich & Poczatek, 2000). Protein phosphatase 2 (PPP2) and Ribosomal protein S6 kinase (RPS6K) are key regulators implicated in the phosphorylation of receptor and SMADs in the TGF-β signaling cascade (Fenton & Gout, 2010; Zolnierowicz, 2000). ID proteins are direct targets of BMP and TGF-β signaling, which serve as essential mediators in biological responses downstream the pathway (Miyazono & Miyazawa, 2002). The cell distribution and coordination of gene expression among the TGF-β genes testify to the significance of this pathway in embryo development.
Furthermore, the expression of many members of the TGF-β superfamily in the endometrium suggests a pivotal role of these genes in the differentiation of the endometrium and the implantation process (Jones et al. 2006). Consequently, the expression of TGF-β genes in preimplantation bovine embryos suggests an important role of these genes in the embryo-uterus connection. Indeed, it has been reported that blastocysts express TGF-β proteins that induce apoptosis of endometrial epithelial cells during implantation (Jones et al. 2006). Collectively, expression of TGF-β signaling genes in the bovine embryo suggests important role for the TGF-β pathway in the preimplantation stage of bovine embryo.
There are also reports that these genes function in maintaining pluripotency in the inner cell mass of bovine blastocysts (Pant & Keefer, 2009). Koide et al. (2009) demonstrated that overactivity of BMP4 signaling led to excessive apoptosis in early mammalian embryo development. Also, La Rosa et al. (2011) reported that supplementation of BMP4 to culture medium of IVF embryos decreased blastocyst production and concluded that a balanced BMP signaling activity is required for proper preimplantation development of cattle embryos.
The ID proteins function as key regulators of development by stimulating and maintaining proliferation and to preventing premature differentiation (Yokota & Mori, 2002), and are known to be regulated by other members of the TGF-β pathway such as BMPs (Hogg et al. 2010). BMPER is a BMP binding endothelial regulator and has been reported to modulate BMP4 signaling in endothelial cell differentiation and angiogenesis (Heinke et al. 2008; Moser et al. 2003).
Although the specific roles in bovine embryo development of differentially expressed genes are unknown, they have critical functions in the TGF-β signaling. For example, SMAD2 belongs to the SMAD family of proteins, which are transducers of TGF-β signal from the cell surface to the nucleus and transcription factors mediating the expression of target genes in the TGF-β cascade (Heldin et al. 1997; Massague et al. 2005). SMAD proteins are required for pluripotency maintenance of the inner cell mass in mouse blastocysts (James et al. 2005).
In a genome-wide association study, a SNP associated with fertilization rate was located within 50 Kb distance of ID3 (Huang et al. 2010a). Although the molecular regulation of fertilization success is not fully understood, maternal genome activity and oocyte quality appear to have critical roles in embryogenesis (Marteil et al. 2009; Stitzel & Seydoux, 2007). Recently, Hogg et al. (2010) observed that four ID isoforms (ID1-4) were expressed across the ovine ovarian follicle development and possibly regulated by TGF-β signaling via SMADs, and suggested mechanistic roles of the ID proteins in mammalian oocyte development. Furthermore, ID proteins are key regulators for many cellular processes, such as cell proliferation, differentiation, and cell cycle progression, which in turn are required for oocyte maturation, oocyte-to-embryo transition, and embryogenesis (Norton, 2000; Stitzel & Seydoux, 2007).
Indeed, it has been acknowledged that blastocyst yield can be affected by intrinsic oocyte quality (Rizos et al. 2002), and the involvement of BMP4 in ovarian function has been extensively reported (Shimasaki et al. 1999). The spatiotemporal expression of BMP4 signaling in follicle development has been broadly observed across different species including human, rat, bovine, swine, and zebrafish (Fatehi et al. 2005; Li & Ge, 2011; Nilsson & Skinner, 2003; Shimizu et al. 2004; Tanwar & McFarlane, 2011). Functional studies have also shown that BMP4 suppresses bovine granulose cell apoptosis and promotes follicle survival and development in rats (Kayamori et al. 2009; Nilsson & Skinner, 2003).
Nevertheless, even though the TGF-β signaling pathway was known to play a crucial role in ovarian and embryonic development, it had not been established whether the balance of expression level of various genes from this pathway is needed for proper preimplantation development of IVF embryos. Furthermore, there was limited information on the extent of contributions of maternal and embryonic genomes to the survival of the developing embryo. More importantly, no indication existed that variations of the maternal genotype in regard to the TGF-β genes had an impact on fertility rates. Identifying genetic factors that show association with fertility would enable selection or breeding programs that reduce the frequency of deleterious alleles at these loci by marker- or gene-assisted selection, preventing further decline or even improving reproductive status of the global dairy herd. In this regard, a plurality of or multiple genes are likely more reliable than a single gene or SNP in predicting high fertility or enhanced embryo survival.